Co-Occurrence of Aflatoxin B1, Zearalenone and Ochratoxin A in Feed and Feed Materials in Central Italy from 2018 to 2022.

Mycotoxin contamination of feed and feed materials represent a serious health hazard. This study details the occurrence of aflatoxin B1 (AFB1), zearalenone (ZEN) and ochratoxin A (OTA) in 826 feed and 617 feed material samples, collected in two Italian Regions (Umbria and Marche) from 2018 to 2022 analyzed using a UPLC-FLD platform. The developed method was validated and accredited (ISO/IEC 17025) with satisfactory accuracy and precision data obtained in repeatability and intralaboratory reproducibility conditions. Feed had a higher incidence of contaminated samples (26%) with respect to feed materials (6%). AFB1 was found up to 0.1045 mg/kg in cattle feeds and 0.1234 mg/kg in maize; ZEN was detected up to 6.420 mg/kg in sheep feed while OTA was rarely reported and in lower concentrations (up to 0.085 mg/kg). Co-contamination of at least two mycotoxins was reported in 0.8% of the analyzed samples. The incidence of above maximum content/guidance level samples was 2% for feed and feed materials while almost 3-fold-higher for maize (5.8%) suggesting how mycotoxin contamination can affect some matrices more than others. Obtained data can be useful to improve official monitoring plans and therefore further raise awareness of this issue between agriculture stakeholders, healthcare entities and non-professionals.

Polar pesticides in food of animal origin: interlaboratory validation to evaluate method fitness-for-purpose of official control.

The present work reports on the design, execution and evaluation of results of an interlaboratory validation study aimed at verifying the fitness-for-purpose of a LC-MS/MS method for the detection of polar pesticides in food of animal origin in official control and monitoring programmes. To this scope, five participant laboratories, with relevant expertise, were recruited. After passing a pre-trial test, the participants were asked to analyse test samples of bovine fat, chicken eggs and cow's milk, contaminated with 11 polar pesticides (group A: Aminomethyl phosphonic acid (AMPA), cyanuric acid, ethephon, glyphosate, fosetyl aluminium, 2-hydroxyethyphosphonic acid (HEPA), maleic hydrazide, N-acetyl-glyphosate, group B: N-acetyl glufosinate (NAG), 3-methylphosphinicopropionic acid (MPP) and glufosinate ammonium) at two different levels (0.05 and 0.25 mg/kg-1 and 0.01 and 0.05 mg/kg-1 for group A and B respectively. The method was based on acidified methanol/water extraction followed by dSPE clean up with C18 sorbent. For LC-MS/MS analysis isotopically labelled standards were used for all targeted analytes. With a couple of exceptions, average recoveries ranged from 85% to 110%, with repeatability (RSDr) ranging from 3% to 25%, and reproducibility (RSDR) from 4% to 26%. The assessment by different laboratories provided also insights on key factors impacting method performance characteristics and its implementation by new users.

Preliminary Investigation about Aspergillus spp. Spread in Umbrian Avian Farms.

Among the fungi responsible for deep mycosis, the genus Aspergillus plays a predominant role both in human and veterinary medicine. From a "One Health" perspective, infections by Aspergillus spp. often represent a public health problem linked to specific occupational categories that could have a greater risk of inhaling spores and developing any respiratory disease. This preliminary investigation allowed to acquire information about the spread of Aspergillus spp. in avian livestock of the Umbria region (Central Italy), their sensitivity to antifungals, and the presence of mycotoxins in the considered farms. Environmental, feed, animal, and human samples were collected for mycological investigations; chemical analyses were also performed in feed samples. Moreover, prevalence estimated of the fungal isolates were provided for each individual farm sampled. Direct fungal identification was possible in 298 out of the 559 total samples; 162 of the samples were positive for Aspergillus spp. Mycotoxins were detected in 5 out of the 21 feed samples collected. All the aspergilli tested for antifungal susceptibility were resistant to fluconazole. The results obtained show how much the genus Aspergillus is widespread in the investigated farms; therefore, the poultry livestock represents a favorable environment for the maintenance and spread of fungal spores and their potential transmission to animals and humans.

The Current Status of Analytical Methods Applied to the Determination of Polar Pesticides in Food of Animal Origin: A Brief Review.

The use of high polar pesticides such as glyphosate and metabolites has increased due to their low cost, low persistence in the environment and high effectiveness. The use of glyphosate is currently permitted in the European Union until 15 December 2022. However, the possible toxic effects on human health and the environment are under debate. Their widespread application on various crops might lead to residues in food intended for animal consumption. For this reason, the Commission, implementing Regulation (EU) 2021/601, recommends the analyses of polar pesticides, not only in matrices of plant origin, but also in those of animal origin such as fat, liver, milk and eggs throughout the years 2022, 2023 and 2024. The determination of polar pesticides is hampered by their chemical nature, which poses challenges both in the instrumental detection (poor column retention, low molecular weight MS/MS fragments, etc.) and in the management of matrix effects, which may vary significantly from matrix to matrix within the same food commodity group. For these reasons, nowadays, there is a limited number of methods for the detection of polar pesticides in food of animal origin. This brief review discusses the different approaches for the simultaneous determination of polar pesticides in food of animal origin using both chromatographic and non-chromatographic techniques.

Monitoring Alternaria toxins in Italian food to support upcoming regulation.

The collection of occurrence data on Alternaria toxins in food and feed across the European countries is required since 2012 by the European Commission, endorsing the relevant scientific opinion by the EFSA CONTAM Panel. Within this framework, occurrence data for Alternaria toxins (Alternariol, Alternariol monomethyl ether, Tenuazonic acid, Tentoxin, and Altenuene) in 97 samples of cereal foods, tomato products, and sunflower seeds have been provided as requested by the Italian national monitoring programme (years 2017-2020). To this purpose, an LC-MS/MS method was set up and validated, obtaining fit for purpose sensitivity, recoveries (70-120%), repeatability (≤20%) and within laboratory reproducibility (≤26%). Occurrence data showed that oilseeds were the most contaminated food group with levels of Tenuazonic acid up to 16752 µg/kg and Tentoxin up to 570 µg/kg, whereas for the other mycotoxin/commodities combinations, the percentage of left censored data (below the limit of quantification) ranged from 74 to 100%.

Undertaking a New Regulatory Challenge: Monitoring of Ergot Alkaloids in Italian Food Commodities.

The present manuscript reports on monitoring data of 12 ergot alkaloids (EAs) in cereal and cereal-derived products, collected in Italy over the period 2017-2020, for official control purposes under the edge of the Commission Recommendation 2012/154/EU on the monitoring of the presence of EAs in feed and food. To these purposes, an LC-MS/MS method was set up and applied, after in-house verification of its analytical performance. Besides satisfactory recoveries and precision, the method's quantification limits proved suitable to assess the compliance of cereals and cereal-based foods with the recently issued EU maximum permitted levels (Commission Regulation 2021/1399/EU). The validity of the generated data was also evaluated through the adoption of four proficiency tests, from which acceptable z-score values (-2 ≤ z ≤ 2) were obtained. The method was then applied to analyse a total of 67 samples, collected in Italy over the period 2017-2020. The samples consisted of 18 cereal grains, 16 flours (14 of wheat and 2 of spelt) and 31 other types of cereals derivatives (including 9 for infants). Overall, the EAs analysis returned a high percentage of left-censored data (>86%). Among the positive samples, the highest contamination levels, up to 94.2 µg/kg, were found for ergocristine (12% incidence), followed by ergocristinine (7% incidence) with levels of up to 48.3 µg/kg.

A Study of the Occurrence of Aflatoxin M1 in Milk Supply Chain over a Seven-Year Period (2014-2020): Human Exposure Assessment and Risk Characterization in the Population of Central Italy.

Aflatoxin food contamination represents a rising global issue that will continue to increase due to climate change. Aflatoxin M1 (AFM1) is of high concern for the whole dairy industry. In light of AFM1's harmful potential, a human health exposure assessment and risk characterization were performed for all age populations of central Italy with regard to milk and cheese consumption by means of the margin of exposure (MOE). In total, 16,934 cow and ewe's milk samples were collected from 2014 to 2020 and analyzed by an enzyme-linked immunosorbent assay (ELISA) screening method, confirmed by high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). The average concentration of AFM1 in cow's milk ranged from 0.009 to 0.015 µg/kg, while in ewe's milk, the average concentration ranged from 0.009 to 0.013 µg/kg. The average amount of AFM1 exposure ranged from 0.00005 to 0.00195 g/kg bw/day, with the main contributor represented by drinking milk, followed by the consumption of soft cheeses. A high level of public health concern related to the youngest consumers has arisen from risk characterizations highlighting the need for constant monitoring of AFM1's occurrence in milk by inspection authorities, alongside regular updates with regard to exposure assessments.

Incidence of ionophore and non-ionophore anticoccidials residues in poultry meat and eggs and their risk characterization.

A multi-residue method was applied to investigate the incidence and the concentration of ionophores and non-ionophore anticoccidials residues in poultry meat and hen eggs for the three-year period 2017-2019 in Italy. The risk related to the ingestion of such molecules was also characterized for the entire population. The average incidences of positive samples ranged from 1.35 to 9.45% while the maximum average concentration was of 4.28 µg/kg for nonionophore molecules. No uncompliant sample was recorded. The overall risk characterization related to the intake of anticoccidials trought chicken meat and eggs reveal a minor concern for consumers of all age. However, the monitoring of coccidiostates residues through official control activity in poultry meat and egg is crucial and it should be continuously conducted to ensure safety of such products and safeguard consumers̛ health.

Critical Comparison of Analytical Performances of Two Immunoassay Methods for Rapid Detection of Aflatoxin M1 in Milk.

Aflatoxin B1 (AFB1) is a secondary metabolite produced by some Aspergillus spp. fungi affecting many crops and feed materials. Aflatoxin M1 (AFM1), the 4-hydroxylated metabolite of AFB1, is the main AFB1-related compound present in milk, and it is categorized by the International Agency for Research on Cancer (IARC) as a "group 1 human carcinogen". The aim of this work was to evaluate and compare the analytical performances of two commercial immunoassays widely applied for the detection of AFM1 in milk, namely strip test immunoassay and enzyme linked immunosorbent assay (ELISA). Assay validation included samples at AFM1 levels of 25, 50, 75 ng/kg and blank samples (AFM1 < 0.5 ng/kg). With respect to a screening target concentration (STC) of 50 ng/kg the two assays showed cut-off values of 37.7 ng/kg and 47.5 ng/kg for strip test and ELISA, respectively, a false suspect rate for blanks <0.1% (for both assays) and a false negative rate for samples containing AFM1 at levels higher than STC, of 0.4% (for both assays). The intermediate precision (RSDip) was <32% for the strip test and <15% for the ELISA. Method verification through long-term intra-laboratory quality control (QC) measurements confirmed the results from the validation study. Furthermore, a satisfactory correlation of the results obtained with both immunoassays and the AOAC Official Method 2000.08 was obtained for the analysis of cow milk samples naturally contaminated with AFM1 at levels within "not detected" (< 0.5 ng/kg) and 50 ng/kg. Finally, the extension of the scope of the strip test method to goat and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation.

Evaluation of Aflatoxin M1 enrichment factor in different cow milk cheese hardness category.

Aflatoxin M1 (AFM1) is a hepatocarcinogenic and genotoxic derivative of aflatoxin B1 excreted into milk after ingestion of feed contaminated by Aspergillus genus fungi. Because of the important role of dairy products, especially cow cheese, in the human diet, there is great concern about the presence of AFM1 in this food category. EC Regulation No. 1881/2006 establishes the importance of the enrichment factor (EF), an essential parameter that must be defined in order to evaluate the maximum level of the toxin in cheese aiming to ensure that cheese has been produced from compliant milk. The Italian Ministry of Health has established two provisional AFM1 EFs (5.5 and 3.0) to be applied to as many cheese categories (hard and soft), defined according to the moisture content on a fat free basis (MFFB) classification. Two experimental productions of Primosale and Fior di Latte cheese, both belonging to the soft cheese category, showed an EF of 4.1 and 2.9 respectively. Data in literature also suggest that the EF attribution based on the current categorization may need reconsideration.

Nutritional Value and Contaminant Risk Assessment of Some Commercially Important Fishes and Crawfish of Lake Trasimeno, Italy.

The aim of our study was to describe the balance between health benefits and risks associated with the consumption of crawfish and nine fish species from lake Trasimeno. We thus determined both fatty acid profiles (particularly, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids) and chemical pollutants (some polychlorinated biphenyls, pesticides, and heavy metals) in fish muscle tissues. The contents of all fatty acids varied significantly among species. Sand smelt, carp, and tench, which have a high fat content, contained considerable amounts of EPA and DHA; lean fish, like perch, pike, and largemouth bass, which have relatively high percentages of the predominant n-3 fatty acids EPA and DHA, showed lower amounts of these fatty acids because of their low lipid contents. Some species contributed strongly to the Dietary Reference Intake (RDI) of EPA and DHA. The contribution of lean fish to the RDI of EPA and DHA was more limited. The concentrations of all contaminants in fish muscle tissues were lower than the regulatory limits, demonstrating the safety of the environmental conditions of the lake. The contribution to health-based reference values and benefit-risk quotients indicated that the health benefits of consumption of fish from lake Trasimeno outweigh the potential risks.

Rapid and reliable detection of glyphosate in pome fruits, berries, pulses and cereals by flow injection - Mass spectrometry.

A flow injection - mass spectrometry method for rapid glyphosate detection in food commodities was developed and validated. The sample preparation protocol included a simple and rapid extract purification step through polymeric solid phase extraction cartridges followed by addition of isotopically labeled glyphosate to the final test sample. The optimized method was subjected to intra-laboratory validation (spiking range 0.5-100 mg/kg) in chickpeas, grapes and apples, as representatives of three different commodity groups as defined in SANTE/11813/2017 guidelines. Recoveries were in the range 60-111%, repeatability and within laboratory reproducibility were ≤17%.The trueness of the results generated with the developed method was evaluated by analysis of a set of incurred chickpea and wheat samples (glyphosate range 0.5-36 mg/kg) and comparison with the reference method (Quick Polar Pesticides Method), confirming the method fitness-for-purpose of rapid compliance testing.

Occurrence and Residue Concentration of Coccidiostats in Feed and Food of Animal Origin; Human Exposure Assessment.

Occurring central Italy, 262 unmedicated feed samples and 353 samples of animal tissues and eggs are tested for coccidiostats between 2012 and 2017. A validated multi-residue HPLC-MS/MS method is applied for the simultaneous determination of the 11 coccidiostats licensed in the EU. The dietary exposure to coccidiostats through poultry meat and eggs is calculated for high consumers, and the contribution to acceptable daily intake of coccidiostats is evaluated. The occurrence of positive feed samples ranges from 17.2% in 2012 to 28.3% in 2017, with an average percentage of positive samples of 25%, while 3.8% of feed samples are non-compliant with a concentration ranging from 0.015 mg/kg for diclazuril to 56 mg/kg for narasin. Positive samples of animal tissues, on average, are 34.7%, fully compliant, while 16% of eggs are positive and violative residues are found in 2%. These noncompliant samples show a concentration varying from 2.4 µg/kg to 1002 µg/kg. The contribution of poultry meat and egg consumption to the acceptable daily intake of each coccidiostat is below 1%, highlighting a low direct risk to public health.

Multimycotoxin Analysis by LC-MS/MS in Cereal Food and Feed: Comparison of Different Approaches for Extraction, Purification, and Calibration.

Twelve different approaches commonly used for the simultaneous LC tandem MS (MS/MS) determination of mycotoxins (deoxynivalenol, aflatoxins, ochratoxin A, T-2 and HT-2 toxins, fumonisins, and zearalenone) were tested in cereals and feed materials. They comprised different extraction solvents, types of cleanup [solid-phase extraction, QuEChERS, and immunoaffinity (IMA)], and calibration approaches (external or matrix-matched). The percentage of mycotoxins with acceptable recovery, according to Regulation (EC) No. 401/2006, ranged from 9 to 100%. The approach giving the highest percentage of acceptable results was selected and further tested for corn, rice, and feed spiked at three different mycotoxin levels (low, medium, and high). The method is based on extraction with MeOH-water (70 + 30, v/v) and cleanup with two multiantibody IMA columns. For corn and rice spiked at low mycotoxin levels, a significant matrix effect was observed and was compensated by using 13C calibration. At higher mycotoxin levels (medium and high), matrix effects were negligible as no significant differences were observed for the majority of recovery results calculated by 13C calibration and external calibration. Although the proposed method still needs improvement in terms of accuracy and, to a lesser extent, precision, it was successfully tested with four proficiency tests in buckwheat, corn, rice, and feed, giving acceptable z-scores for 97% (34 out of 35) of results.

MycoKey Round Table Discussions of Future Directions in Research on Chemical Detection Methods, Genetics and Biodiversity of Mycotoxins.

MycoKey, an EU-funded Horizon 2020 project, includes a series of "Roundtable Discussions" to gather information on trending research areas in the field of mycotoxicology. This paper includes summaries of the Roundtable Discussions on Chemical Detection and Monitoring of mycotoxins and on the role of genetics and biodiversity in mycotoxin production. Discussions were managed by using the nominal group discussion technique, which generates numerous ideas and provides a ranking for those identified as the most important. Four questions were posed for each research area, as well as two questions that were common to both discussions. Test kits, usually antibody based, were one major focus of the discussions at the Chemical Detection and Monitoring roundtable because of their many favorable features, e.g., cost, speed and ease of use. The second area of focus for this roundtable was multi-mycotoxin detection protocols and the challenges still to be met to enable these protocols to become methods of choice for regulated mycotoxins. For the genetic and biodiversity group, both the depth and the breadth of trending research areas were notable. For some areas, e.g., microbiome studies, the suggested research questions were primarily of a descriptive nature. In other areas, multiple experimental approaches, e.g., transcriptomics, proteomics, RNAi and gene deletions, are needed to understand the regulation of toxin production and mechanisms underlying successful biological controls. Answers to the research questions will provide starting points for developing acceptable prevention and remediation processes. Forging a partnership between scientists and appropriately-placed communications experts was recognized by both groups as an essential step to communicating risks, while retaining overall confidence in the safety of the food supply and the integrity of the food production chain.

Development and validation of LC-MS/MS method for the determination of Ochratoxin A and its metabolite Ochratoxin α in poultry tissues and eggs.

The objective of this study was to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of Ochratoxin A (OTA) and Ochratoxin α (OTα) in poultry tissues and eggs. The two toxins were extracted by a mixture of acetonitrile/water, purified with a reversed phase C18 solid phase extraction column (SPE) and determined by LC-MS/MS. The LC-MS/MS method performances were evaluated in terms of linearity in solvent and in matrix (ranged from 0.5 to 15.10 µg L-1 for OTA and from 0.60 to 17.85 µg L-1 for OTα), limit of detection (LOD), limit of quantitation (LOQ), specificity, accuracy and precision in repeatability conditions. Recovery experiments were performed by spiking poultry liver, kidney, muscle and eggs around 1 µg kg-1 and 10 µg kg-1. LODs were 0.27 and 0.26 µg kg-1 while LOQs were fixed at 1.0 and 1.2 µg kg-1 for OTA and OTα, respectively. Main recoveries for OTA ranged from 82 to 109% and for OTα ranged from 55 to 89%. The values of within-laboratory relative standard deviation (RSDr) were equal to or below 20%. Considering the results obtained and that all analytical performance criteria were fulfilled, the new extraction and purification method developed for OTA and OTα determination in animal tissues and eggs was found appropriate for control laboratories and research activities designed to ensure food safety.

Rapid determination of residues of pesticides in honey by µGC-ECD and GC-MS/MS: Method validation and estimation of measurement uncertainty according to document No. SANCO/12571/2013.

A simple and straightforward method for simultaneous determination of residues of 13 pesticides in honey samples (acrinathrin, bifenthrin, bromopropylate, cyhalothrin-lambda, cypermethrin, chlorfenvinphos, chlorpyrifos, coumaphos, deltamethrin, fluvalinate-tau, malathion, permethrin and tetradifon) from different pesticide classes has been developed and validated. The analytical method provides dissolution of honey in water and an extraction of pesticide residues by n-Hexane followed by clean-up on a Florisil SPE column. The extract was evaporated and taken up by a solution of an injection internal standard (I-IS), ethion, and finally analyzed by capillary gas chromatography with electron capture detection (GC-µECD). Identification for qualitative purpose was conducted by gas chromatography with triple quadrupole mass spectrometer (GC-MS/MS). A matrix-matched calibration curve was performed for quantitative purposes by plotting the area ratio (analyte/I-IS) against concentration using a GC-µECD instrument. According to document No. SANCO/12571/2013, the method was validated by testing the following parameters: linearity, matrix effect, specificity, precision, trueness (bias) and measurement uncertainty. The analytical process was validated analyzing blank honey samples spiked at levels equal to and greater than 0.010 mg/kg (limit of quantification). All parameters were satisfactorily compared with the values established by document No. SANCO/12571/2013. The analytical performance was verified by participating in eight multi-residue proficiency tests organized by BIPEA, obtaining satisfactory z-scores in all 70 determinations. Measurement uncertainty was estimated according to the top-down approaches described in Appendix C of the SANCO document using the within-laboratory reproducibility relative standard deviation combined with laboratory bias using the proficiency test data.

Deadly outbreak of iron storage disease (ISD) in Italian birds of the family Turdidae.

A widespread deadly outbreak occurred in captive birds belonging to the family Turdidae in Italy. The present study was performed on 46 dead birds coming from 3 small decoy-bird breeders in central Italy. Only Turdus pilaris, Turdus iliacus, Turdus philomelos and Turdus merula were affected. No other species of bird held by these breeders died. A change of diet before the hunting season was reported from all breeders. Full necropsy of the animals and histological investigations of representative tissue samples were performed. Microscopical examination showed marked iron deposits in liver samples. Bacteriological investigations and molecular analysis to exclude bacterial and viral diseases were carried out. Contamination of food pellet samples by mycotoxins and analysis to detect heavy metal contaminants in food pellet samples were considered. An interesting result was the high iron content found in food pellets. It was higher than that considered suitable for birds, especially for species susceptible to development iron storage disease (ISD). Taken together, the results suggested an outbreak of ISD caused by the high iron content of food given to the birds before the hunting season. The high mortality recorded only in species belonging to the family Turdidae suggests a genetic predisposition in the affected birds.

Temporal trends of PCBs in feed and dietary influence in farmed rainbow trout (Oncorhynchus mykiss).

As a rainbow trout producer, Italy is accounted as fifth in the world and second in continental Europe. In this study, the levels of the eighteen PCBs in feed and in trout, showed a statistical significant difference (p<0.01) throughout the years, with a declining trend from 2005 to 2010. This trend shows effectively that quality and safety of trout feeds has greatly improved during the last years and, as a consequence, also the PCBs values in muscle trout, showed a decreasing trend. Moreover, feed Σ18PCBs showed a statistical significant difference (p<0.01) among the analysed brands and was positively correlated (p<0.01 and r=0.451) with the rainbow trout muscle Σ18PCBs. These results showed that the presence of PCBs in trout muscle is directly linked to the chemical quality of aquaculture feed. The most commonly detected PCBs congeners were congeners PCB 153 and PCB 138 in all the three compared brands.

Biosensor-based screening method for the detection of aflatoxins B1-G1.

Aflatoxins are extremely toxic metabolites from Aspergillus species that can adulterate a wide range of human foodstuff. Herein, we propose a novel assay designed as an analytical test for aflatoxin B1 and G1 (AFB1 and AFG1, respectively) that could represent an alternative screening technique for this class of mycotoxins. The approach for the determination of these toxins is based on surface plasmon resonance using neutrophil porcine elastase as a "bait" for these aflatoxins. The selection and optimization of the analytical procedure involved a preliminary investigation on the type of inhibition by AFB1: the level of the protease inhibition exerted by AFB1 depended upon the incubation time and the concentration of the binding partners, showing the competitiveness and the reversibility of the inhibition. A posteriori, the nature of the interaction granted a rapid analysis, a single detection test requiring only a few minutes. For the development of the assay, the experimental conditions were evaluated and optimized with both calibration solution and aflatoxin-spiked samples. To apply this method to aflatoxin-contaminated maize, a rapid solid-phase extraction treatment was developed. The proposed assay for AFB1 and AFG1 was validated by comparison with both a chromatographic reference method and a standard enzyme linked immunosorbent assay procedure. This enzyme-based biosensor represents a new approach for the detection of aflatoxins based on the reversible interaction between a blocked macromolecule and a soluble ligand, having the major advantages in the relative rapidity, the reusability of the capturing surface, and low cost per single test.